InteractiveFly: GeneBrief

ALG3, alpha-1,3- mannosyltransferase: Biological Overview | References

Drosophila tumor suppressor genes have revealed molecular pathways that control tissue growth, but mechanisms that regulate mitogenic signaling are far from understood. This study reports that the Drosophila TSG tumorous imaginal discs (tid), whose phenotypes were previously attributed to mutations in a DnaJ-like chaperone, are in fact driven by the loss of the N-linked glycosylation pathway component ALG3. tid/alg3 imaginal discs display tissue growth and architecture defects that share characteristics of both neoplastic and hyperplastic mutants. Tumorous growth is driven by inhibited Hippo signaling, induced by excess Jun N-terminal kinase (JNK) activity. Ectopic JNK activation is caused by aberrant glycosylation of a single protein, the fly tumor necrosis factor (TNF) receptor homolog, which results in increased binding to the continually circulating TNF. These results suggest that N-linked glycosylation sets the threshold of TNF receptor signaling by modifying ligand-receptor interactions and that cells may alter this modification to respond appropriately to physiological cues (de Vreede, 2018).

Tumorigenesis is ultimately driven by dysregulated cellular signaling that promotes unchecked proliferation. Proliferation-regulating signaling pathways in animals are therefore normally under tight control, to prevent aberrant growth. The primary mechanism of signaling regulation is limited availability of ligand, although levels of receptor can also be regulated, as can receptor availability on the plasma membrane or even its polarized localization. A full understanding of the mechanisms that limit mitogenic signaling is an important goal of both basic biology and cancer research (de Vreede, 2018).

Major insight into growth regulation has arisen from research in model organisms such as Drosophila melanogaster. For instance, Drosophila studies revealed key steps of receptor tyrosine kinase signaling and uncovered the phenomenon of cell competition. Additional insight into growth regulatory mechanisms has come from the analysis of fly tumor suppressor genes (TSGs). Disruption of a single fly TSG is sufficient to cause overproliferation in epithelial organs of the larva called imaginal discs. Initial genetic screens identified several classes of fly TSGs. The neoplastic TSGs (discs large, lethal giant larvae, and scribble) revealed an intimate link between cell polarity and cell proliferation control, a principle also relevant to human cancers. The hyperplastic TSGs, including hippo, warts, and salvador, uncovered the novel Hippo (Hpo) signal transduction pathway, which is now recognized as a conserved growth control mechanism. Even less prominent Drosophila TSGs such as lethal giant discs have demonstrated important biological concepts (de Vreede, 2018).

One classic Drosophila TSG that remains understudied is tumorous imaginal discs (tid) (Gateff, 1978, Löffler, 1990). Imaginal discs of tid homozygous larvae develop into overgrown masses (Kurzik-Dumke, 1995). Genetic mapping and cytogenetic analyses attributed this phenotype to loss of a conserved molecular chaperone of the DnaJ family (Kurzik-Dumke, 1995). Evidence for a tumor-suppressive role for a mammalian homolog, hTid-1, has been presented (Chen, 2009, Copeland, 2011, Kurzik-Dumke, 2008). However, the exact molecular mechanism through which tid could regulate cell and tissue proliferation remains mysteriou (de Vreede, 2018).

This study reports that the tid gene was cloned incorrectly. Aberrant cell proliferation in the Drosophila mutant arises not from disruptions to the DnaJ homolog but rather to an adjacent gene that encodes the mannosyltransferase ALG3, involved in N-linked glycosylation. Overgrowth in tid/ALG3 mutants is caused by mis-glycosylation of a single transmembrane protein, the Drosophila tumor necrosis factor (TNF) receptor homolog Grindelwald, which results in downstream activation of Jun N-terminal kinase (JNK) and inactivation of the growth-suppressing Hpo pathway. The results suggest that this post-translational modification modulates ligand-receptor affinity in the TNF receptor (TNFR) pathway and thus provides a regulatory mechanism setting a dynamic threshold for JNK-mediated stress signaling and growth control (de Vreede, 2018).

This study has shown that mutations in the classic Drosophila TSG tumorous imaginal discs (tid) disrupt the ALG3 homolog CG4084, altering the lipid-linked biosynthetic pathway that generates oligosaccharides for protein N-linked glycosylation. Although altered glycosylation affects many proteins and can induce a unfolded protein response (UPR), this study finds that the growth control phenotype of Alg3 can be ascribed to a single target and a single mechanism. This target is the Drosophila TNFR homolog, whose proper modification at a single extracellular site is required to prevent inappropriate TNF binding, subsequent JNK activation, and downstream Yki-driven overproliferation. It is postulated that N-glycosylation can act as a mechanism to modulate JNK signaling in response to cellular stresses (de Vreede, 2018).

The alg3 mutations were originally identified for their overgrowth phenotype in imaginal discs (Kurzik-Dumke, 1992). Like most other Drosophila TSGs, this phenotype is caused by changes in Hpo-regulated Yki activation, but alg3 mutants differ in both upstream regulation and downstream targets. Mutations in core Hpo signaling components result in rapid proliferation of disc cells, while the slow growth of alg3 mutant tissue resembles that of the neoplastic TSGs. Nonetheless, the STAT pathway, which is a major mitogenic effector in neoplastic mutants, is not elevated in alg3 tissue. Upstream, JNK-dependent Yki activity is seen in both alg3 and neoplastic mutants. However, JNK activation in neoplastic mutants has been suggested to occur either through ligand-independent Grnd activation caused by alteration to apicobasal polarity or through Grnd-independent mechanisms. In alg3 mutants, polarity is intact and overgrowth entirely relies on a Grnd-Egr axis, specifically the increased sensitivity of misglycosylated Grnd for endocrine Egr. Thus, TNFR signaling induced by altered N-glycosylation seems to define distinct consequences for downstream Hpo-mediated growth control (de Vreede, 2018).

While this study has not tested biochemical affinities directly, the data are consistent with a model where TNF-binding properties are directly regulated by glycosylation of TNFR. Partial or complete removal of the glycan at N63, within the ligand-binding domain of Grnd, leads to an increase of bound Egr, indicating that N-glycosylation normally limits Grnd engagement and downstream signaling. In Drosophila larvae, Egr is continuously transcribed in the fat body for secretion into the hemolymph, bathing Grnd-expressing tissues, including imaginal discs and IPCs in ligand. The results suggest that proper N-glycosylation of Grnd sets a threshold that prevents tonic signaling in these and other tissues under normal circumstances. This raises the intriguing possibility that cell-autonomous changes in N-glycosylation, perhaps induced by stress inputs, could modulate ligand affinity, allowing a rapid and local response to this endocrine signal under different physiological conditions (de Vreede, 2018).

The modulation of Grnd ligand binding suggested here echoes the regulation of Notch by the glycosyltransferase Fringe. However, the obligate role of Alg3 in all N-glycan synthesis is fundamentally distinct from Fringe's substrate-specific elaboration of a particular O-glycan. In the case of Notch, the specific sugar residues added by Fringe alter receptor selectivity for one ligand over another. Since either aberrant or absent Grnd N-glycosylation results in increased ligand binding and ectopic signaling, evidence for specific glycan structures in modulating the ligand-receptor interface does not currently exist. Whether the glycan could provide a simple steric obstacle to ligand binding or may regulate it through more complex interactions will await structural studies (de Vreede, 2018).

Grnd shows strong homology to vertebrate TNFR family members in its extracellular TNF-binding domain, although downstream signaling in the fly acts mainly through JNK, in contrast to mammalian homologs that also signal through nuclear factor κB (NF-κB), p38, and caspases. Among the 29 mammalian TNFR superfamily members, at least seven have predicted N-glycosylation sites in their extracellular domains. Several of these sites have been studied, and their proposed roles vary from promoting signaling to inhibiting it or being functionally neutral. The current results motivate analyses of the receptors BCMA and DR4, which are closely related to Grnd and whose predicted N-glycosylation sites each lie in an analogous location within the ligand-binding domain (Andersen, 2015; de Vreede, 2018 and references therein).

The data presented above, which highlight a new mechanism for restraining TNF signaling, hint at pathogenic mechanisms for several human diseases. Altered glycosylation is emerging as a frequent hallmark of cancer, in which JNK signaling is increasingly implicated. Moreover, mutations in the extracellular domain of human TNFR1, including predicted N-glycosylation sites, can cause the autoinflammatory disease TRAPS (TNFR-associated periodic syndrome). Because the erroneous activation of Grnd in alg3 mutants is akin to an autoinflammatory response, defective N-glycosylation could be an additional mechanism for hyperactive TNFR1 signaling. Finally, mutations in N-glycosylation pathway enzymes including Alg3 result in recessive genetic diseases called type I congenital disorders of glycosylation (CDG-I). CDG patients exhibit a variety of poorly characterized symptoms associated with multiple organs, and the etiology of CDG is largely unknown. The finding of altered inflammatory TNFR/JNK signaling in analogous fly mutants provides a new avenue to investigate (de Vreede, 2018).

Search PubMed for articles about Drosophila

Andersen, D. S., Colombani, J., Palmerini, V., Chakrabandhu, K., Boone, E., Rothlisberger, M., Toggweiler, J., Basler, K., Mapelli, M., Hueber, A. O. and Leopold, P. (2015). The Drosophila TNF receptor Grindelwald couples loss of cell polarity and neoplastic growth. Nature 522(7557): 482-486. PubMed ID: 25874673

Chen, C. Y., Chiou, S. H., Huang, C. Y., Jan, C. I., Lin, S. C., Hu, W. Y., Chou, S. H., Liu, C. J. and Lo, J. F. (2009). Tid1 functions as a tumour suppressor in head and neck squamous cell carcinoma. J Pathol 219(3): 347-355. PubMed ID: 19681071

Copeland, E., Balgobin, S., Lee, C. M. and Rozakis-Adcock, M. (2011). hTID-1 defines a novel regulator of c-Met Receptor signaling in renal cell carcinomas. Oncogene 30(19): 2252-2263. PubMed ID: 21242965

de Vreede, G., Morrison, H. A., Houser, A. M., Boileau, R. M., Andersen, D., Colombani, J. and Bilder, D. (2018). A Drosophila tumor suppressor gene prevents tonic TNF signaling through receptor N-glycosylation. Dev Cell 45(5): 595-605 PubMed ID: 29870719

Gateff, E. (1978). Malignant neoplasms of genetic origin in Drosophila melanogaster. Science 200(4349): 1448-1459. PubMed ID: 96525

Kurzik-Dumke, U., Phannavong, B., Gundacker, D. and Gateff, E. (1992). Genetic, cytogenetic and developmental analysis of the Drosophila melanogaster tumor suppressor gene lethal(2)tumorous imaginal discs (1(2)tid). Differentiation 51(2): 91-104. PubMed ID: 1473626

Kurzik-Dumke, U., Gundacker, D., Renthrop, M. and Gateff, E. (1995). Tumor suppression in Drosophila is causally related to the function of the lethal(2) tumorous imaginal discs gene, a dnaJ homolog. Dev Genet 16(1): 64-76. PubMed ID: 7758246

Kurzik-Dumke, U., Horner, M., Czaja, J., Nicotra, M. R., Simiantonaki, N., Koslowski, M. and Natali, P. G. (2008). Progression of colorectal cancers correlates with overexpression and loss of polarization of expression of the htid-1 tumor suppressor. Int J Mol Med 21(1): 19-31. PubMed ID: 18097612

Linnoila, J., Wang, Y., Yao, Y. and Wang, Z. Z. (2008). A mammalian homolog of Drosophila tumorous imaginal discs, Tid1, mediates agrin signaling at the neuromuscular junction. Neuron 60(4): 625-641. PubMed ID: 19038220

Rozakis-Adcock, M. (2011). hTID-1 defines a novel regulator of c-Met Receptor signaling in renal cell carcinomas. Oncogene 30(19): 2252-2263. PubMed ID: 21242965

date revised: 19 September 2018

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